Immunological hormone assay techniques rely upon the ability of a given hormone to act as an antigen in a given immunological reaction.
Based on this principle, radioimmunoassay techniques have recently been developed in which an unknown amount of unlabeled (cold) hormone to be determined is incubated -- in the presence of a known amount of the same labeled hormone -- with a suitable amount of a specific antiserum. Thus, an unknown amount of hormone can be easily estimated from the radioactivity count of the antigen-antibody complex through comparison with a standard curve previously constructed using known amounts of the specific anti-serum, cold hormone and labeled hormone, both cold and labeled hormones having proportionally concurred (that is, in proportion to their respective amounts) in the formation of the said antigen-antibody complex. Obviously, the radioactivity count of said antigen-antibody complex is inversely proportional to the amount of cold hormone added to the reaction mixture, the antiserum and labeled hormone amounts being left unchanged.
Unfortunately, the radioimmunoassay techniques have not been applicable as yet to the determination of human chorionic gonadotropin (HCG) in the presence of comparable amounts of luteinizing hormone (LH), because of the extreme difficulty of obtaining a specific antiserum for either hormone.
On the other hand, the need of reliable means for performing such determination is particularly recognized in this field. In fact, it is known that such reliable means would permit, among other things, pregnancy to be quickly ascertained (within a few days from conception) and choriocarcinoma as well as any HCG-producing tumors to be detected and controlled.
Therefore, the enormous clinical utility of such specific antiserum in diagnosing and controlling said tumors, as well as in ascertaining pregnancy at an early stage, is clearly apparent.
Very recently Ross et al., "Structure-activity relationships of protein and polypeptide hormones," Part 1 -- Reports-Proceedings of the Second International Symposium Liege, Sept. 28-Oct. 1, 1971; pages 153-7; M. Margoulies and F. C. Greenwood Editors; published by Excerpta Medica (1971) have tested antisera raised against .alpha. and .beta. subunits of HCG (hereinafter referred to as HCG-.alpha. and HCG-.beta., respectively) in radioimmunological reactions and found that the antiserum raised against HCG-.beta. has a higher specificity.
From a graphic display in which, among other curves, two inhibition curves of the radioimmunological reaction between anti-HCG-.beta. serum and labeled HCG-.beta. appear, which have been constructed using HCG and human pituitary LH, respectively, it can be noted that the said two inhibition curves differ in slope.
Urinary LH was not tested by Ross. Since it is known that urinary LH and pituitary LH have generally different immunological behavior, probably due to their dissimilar chemical structures and antigenic sites, no suggestion can be drawn from Ross on whether and to what extent inhibition will be produced by urinary LH on the anti-HCG-.beta. serum plus labeled HCG-.beta. system.
Furthermore, from the quantitative point of view, the two curves reported by Ross are almost coincident at least as far as the portions corresponding to low levels of cold hormone are concerned (that is, up to about 250-300 m I.U./ml).
In this respect, it should be noted that the above mentioned low hormone levels are just the levels at which a discrimination of HCG hormone from LH is required, since the physiological LH levels is urine may at most amount to 150 m I.U./ml. Consequently, the technique developed by Ross et al. cannot be used for a standard assay method of determining HCG in the presence of comparable amounts of LH.
As used herein, "m I.U." means the thousandth part of the International Unit adopted by the World Health Organization on the basis of the relevant reference preparation which is the "Second International Standard for HCG" when HCG is concerned or the "Second International Reference Preparation -- HMG" when LH is concerned. Both of the said reference preparations are available from the Biological Standard Department of W.H.O., England.
As used herein "comparable amounts of LH" means amounts of this hormone (as expressed in terms of m I.U./ml) up to about 10 times those of HCG.